The NCCAM Intramural Endocrine laboratory is focused on the effects of selected androgenic (DHEA; dehydroepiandrosterone), estrogenic (phytoestrogens) or other dietary supplements and/or natural products on neoplastic prostate epithelial and stromal cell growth, gene expression, including signaling between stromal and epithelial cells and the intracellular signal transduction pathways. As a precursor to both estrogen and testosterone, DHEA excess may pose a potential cancer risk in hormone-responsive tissues such as the prostate. We have investigated effects of DHEA on epithelial and stromal cells from normal and cancer tissues, grown separately and in co-culture. Prostate cancer epithelial cells exhibit differential responses, to DHEA, depending in part upon their androgen receptor (AR) status. Prostate cancer cells with a mutated AR and are responsive to DHEA treatment as measured by cell proliferation, and expression of prostate specific antigen (PSA), and IGF axis proteins. In contrast, prostate cancer cells, with a normal AR, are minimally responsive. Prostatic primary stromal cells (6S cells) were responsive to androgens, such as dihydrotestosterone (DHT) by increasing secretory IGF-1, whereas DHEA did not produce the same effect. [unreadable] Important mechanisms of DHEA effects on prostate cells were revealed when cancer cells (with the normal AR) were combined in culture with the stromal cells. When cancer cells were grown in the presence of stromal cells, DHEA stimulated epithelial PSA protein levels to that achieved by DHT-induction. DHEA-treated stromal cells were able to metabolize DHEA to androgenic ligands thereby inducing increased epithelial PSA production in these cocultures. (see Arnold et al, 2008 #1)[unreadable] Addition of the cytokine TGFbeta models an immuno-reactive prostatic stroma as is associated with the cancer tissue microenvironment. TGFbeta -1 added to stromal cells increased epithelial PSA protein secretion 2-4 times over DHEA alone and increased gene expression up to 50-fold. Also in cocultures of LAPC-4 + 6S cells, TGFbeta -1 increased metabolism of DHEA to testosterone. These results indicate that in cancer tissues compared to normal prostate, metabolism of DHEA to androgenic ligands, may have increased induction of growth and PSA production. This hypothesis provides a novel unique paradigm in understanding cancer promotion, incorporating the importance of increased steroid hormone metabolism as a promoting factor in prostate cancer. Red Clover contains various isoflavones, including genistein, daidzein biochanin A, and formononetin. Red clover isoflavones decreased PSA protein and gene expression and T metabolism induced by TGFbeta +DHEA in prostate LAPC-4/6S cocultures in a dose dependent manner. In this coculture model of endocrine-immune-paracrine interactions in the prostate, provides a tool for discovery of natural products or traditional medicines that may be involved in stromal-epithelial cell interactions especially altering paracrine hormonal signals. (see Gray et al, 2008) [unreadable] A parallel study in prostate cell signaling explored role of the natural product, lycopene, in modulating DHT-induced intracellular and extracellular stromal signaling and the resulting effects on epithelial cell signal transduction and cell death. Comparative studies of paracrine signaling from reactive versus normal stroma were carried out using normal human prostate epithelial (NPE) cells. In this study, lycopene, in dietary concentrations, reversed DHT effects of prostate stromal 6S cells on induced NPE cell death; decreased 6S cell IGF-I production by reducing AR and beta-catenin nuclear localization; and inhibited IGF-I-stimulated NPE and Prostate epithelial cells (PREC) growth by attenuating IGF-I's effects on serine phosphorylation of Akt and GSK3 beta and tyrosine phosphorylation of GSK3. This study provides possible mechanisms of lycopene prevention of prostate cancer. (See Liu et al, 2008)[unreadable] Stromal paracrine targets are being identified by gene expression microarray analysis (Affymetrix HU133 chips) of DHEA vs androgen (R1881)-treated stromal cells in coculture vs. monoculture with cancer epithelial cells and signaling mechanisms elaborated. Also stromal paracrine mediators were probed using SuperArray Real Time RT-PCR technology. These studies have highlighted important factors released from reactive stroma compared to normal stromal cells. Many prostate stromal genes have been identified and are being characterized as targets for stromal regulation of epithelial cell function, especially targets for intrinsically multi-mechanistic botanical mixtures. [unreadable] To define endocrine mechanisms involved in the cell interactions, hydroxysteroid dehydrogenase (HSD) metabolizing enzymes were targeted by siRNA knockdown in the stromal cells of the cocultures to determine effects of TGFbeta on metabolism of DHEA. [unreadable] DHEA metabolism to estrogens can play an important role in prostate function. ERbeta is an important target in prostate cells for endogenous and exogenous estrogens and plays a role in modulating androgen activity. Steroid hormones cross-talk with Wnt/beta- catenin pathway and may interact with ER beta in prostate cells. The signal transduction pathways involved in DHEA and its estrogenic metabolites were investigated in prostate cancer cells that are AR(-) but ER beta positive. The study offers a new insight of the non-genomic effect of these steroid hormones. These signaling pathways further elaborate intracellular response to steroid hormones in prostate cells.[unreadable] Additional work within this pathway has elaborated the downstream signaling of Wnt and the interactions between adipose differentiation-related protein (ADRP), Dishevelled (Dvl) and Axin complexes and subsequent rapid inactivation of GSK3 beta/Axin complexes. This work may provide molecular mechanisms of occurrence of obesity associated diseases such as cancer, diabetes and structural birth defects by women with prepregnancy obesity.